Method and device for enhancing sensitivity of immunoblotting and immunofixation electrophoresis

Abstract

The invention relates to a method and a device for enhancing immunoblotting and immunoblotting electrophoresis sensitivity. The method comprises the following steps: performing regular stripe standing wave concentration on protein by utilizing regular stripe standing waves generated by superposing surface sound source ultrasonic waves through a gel electrophoresis carrier which is subjected to electrophoretic separation and contains a low-concentration protein molecule band in a direction vertical to electrophoretic separation; and the protein molecules with different densities are pushed to the node or antinode region of the standing wave and are concentrated. The device comprises an area sound source generator operated by the method and an echo carrier plate with an echo plate. An electrophoresis tank for accommodating an echo carrying disc is additionally arranged, so that the electrophoretic separation step and the concentration step are integrated; and a main bearing body is arranged for bearing the electrophoresis tank, a related power supply and a related switch. According to the method and the device, the concentration of the low-abundance protein in the reaction area reaches or is closer to the optimal concentration for antigen-antibody reaction, in the immunoblotting process, the local luminescence or fluorescence peak intensity after membrane transfer meets the condition of reaching a threshold value or above, and the detection rate of the low-abundance target protein is greatly improved.

Description

Technical Field

[0001] This invention belongs to the field of electrophoretic separation technology, and particularly relates to the field of electrophoretic separation in immunoblotting and immunofixation methods, specifically a method and apparatus for enhancing the sensitivity of immunoblotting and immunofixation electrophoresis. Background Technology

[0002] Immunofixation electrophoresis (IFE) is an important laboratory analytical technique that combines the efficient separation capabilities of band electrophoresis with the specificity of immunoprecipitation. It is primarily used to detect and identify abnormal proteins in biological samples (such as serum and urine), playing an irreplaceable role, particularly in the identification and typing of monoclonal immunoglobulins (M proteins). The process of immunofixation electrophoresis can be summarized in three core steps, the final result of which is the formation of specific precipitated bands, thereby enabling the identification of abnormal proteins. These three steps are electrophoretic ionization, immunoreaction, and color development and analysis. Electrophoretic separation involves spotting a patient's serum or urine sample onto an agarose gel plate or polyacrylamide gel plate, followed by the application of an electric field. Various proteins in the sample, due to differences in charge and molecular size, migrate in the gel and are separated into different bands. The immunoreaction involves directly covering the gel with specific antiserum filter paper, according to different lanes. These antisera target different types of heavy chains (such as IgG, IgA, and IgM) and light chains (κ and λ types). If an abnormal monoclonal immunoglobulin is present in a sample, it will bind to the corresponding specific antibody, forming an insoluble antigen-antibody complex that precipitates. Colorimetric analysis involves washing away unbound proteins and then staining the precipitated complex. Monoclonal proteins will show a concentrated, narrow staining band, while normal polyclonal proteins will form a diffuse, light, broad band. By comparing these bands, the type of abnormal protein can be accurately determined.

[0003] Western blotting, a widely used experimental technique in molecular biology, biochemistry, and immunogenetics, has a wide range of applications, covering protein detection and quantification, molecular weight determination, disease diagnosis, basic biomedical research, clinical trials and regulatory testing, single-cell proteomics research, signal transduction mechanism exploration, protein modification analysis, protein-protein interaction research, and drug development. Due to its high specificity and sensitivity, Western blotting plays a crucial role in biomedical research and clinical diagnosis. Western blotting (WB), also known as Western blot, is based on the core concept of SDS-polyacrylamide gel electrophoresis (SDS-PAGE) to effectively separate mixed protein samples based on differences in protein molecular weight. These separated proteins are then transferred to a solid support, such as a nitrocellulose membrane or PVDF membrane, for subsequent processing. To avoid interference from non-specific binding, the transferred membrane is first treated with a blocking solution containing protein (e.g., skim milk powder or bovine serum albumin). Next, a highly specific primary antibody (i.e., an antibody targeting the target protein) binds to the protein on the membrane. Then, a secondary antibody labeled with an enzyme or fluorophore (i.e., an antibody targeting the primary antibody) is introduced for further detection. Finally, the signal generated by the secondary antibody is observed and analyzed using techniques such as chemiluminescence (ECL), fluorescence display, or staining, thereby indirectly confirming the presence and relative abundance of the target protein.

[0004] Both methods require electrophoretic separation based on protein charge, followed by an antigen-antibody reaction before subsequent qualitative or relative quantification of the protein. The antigen-antibody reaction refers to the specific binding reaction between an antigen and its corresponding antibody. The antigen-antibody reaction is characterized by four main properties: specificity, proportionality, reversibility, and phased nature. Proportionality means that a visible reaction between antigen and antibody requires a specific quantitative ratio; a visible reaction only occurs when their concentrations are in an appropriate ratio. The reaction is most thorough and produces the largest and most abundant immune complex precipitate when the antigen-antibody ratio is roughly equal or when there is a slight excess of antigen. However, when the antigen-antibody ratio deviates significantly from the above ratio, a hook effect occurs, rapidly reducing or even eliminating the antigen-antibody complex reaction.

[0005] Currently, in immunofixation electrophoresis and Western blotting techniques, there is a situation where the protein concentration in the individual bands obtained after the electrophoretic separation step is low. The low concentration of protein cannot meet the requirements of the subsequent antigen-antibody reaction ratio, and cannot react sufficiently with the corresponding antibody to form an effective antigen-antibody complex. In Western blotting, the signal peak intensity generated by the low concentration of protein bands is low, below the detection threshold, which leads to the missed detection of low-abundance analytes or their inability to be detected in time, thus affecting subsequent judgment. Summary of the Invention

[0006] In immunoblotting and immunofixation electrophoresis, due to the complexity of sample components, it is not possible to improve the detection rate of specific protein components by concentrating the total sample (high concentrations of the sample components to be detected will clog the channels of agarose gel or polyacrylamide gel, leading to electrophoresis failure). However, traditional electrophoresis methods result in concentrations of the proteins to be detected that are too low after electrophoretic separation to meet the requirements of antigen-antibody ratio. This application provides a method and apparatus to enhance the sensitivity of immunoblotting and immunofixation electrophoresis. The method utilizes a sound wave generating structure and a reflecting device to form a regular striped standing wave. The striped standing wave is perpendicular to the electrophoresis line. In the direction of electrophoretic separation, a gel electrophoresis carrier containing low concentrations of protein molecules, already separated by electrophoresis, is used. Acoustic radiation pressure pushes protein molecules of different densities towards acoustic pressure nodes or antinodes. Protein molecules less dense than the gel medium are pushed towards nodes, while those denser are pushed towards antinodes. Ultimately, the enrichment effect of acoustic radiation pressure increases the concentration of the target protein molecules, bringing the antigen-to-antibody ratio closer to meet the proportionality requirements of antigen-antibody reactions. This allows the target protein molecules to be detected, solving the problem of undetectable low-concentration analytes. The apparatus is designed to ensure the operation of the above method.

[0007] The specific technical solution is as follows: a method for enhancing the sensitivity of immunoblotting and immunofixation electrophoresis, the steps of which include,

[0008] S1: A regular striped standing wave generated by the superposition of ultrasonic waves from two coherent surface sound sources with the same frequency and amplitude in opposite directions.

[0009] S2: Regular stripe standing wave concentration of proteins is achieved by passing the regular stripe standing wave of a gel electrophoresis carrier containing low concentrations of protein molecular bands after electrophoresis separation in a direction perpendicular to the electrophoretic separation direction.

[0010] The pressure of acoustic radiation is used to push protein molecules of different densities toward the nodes or antinodes of a standing wave; protein molecules with a density less than that of the gel medium are pushed toward the antinodes, while protein molecules with a density greater than that of the gel medium are pushed toward the nodes.

[0011] Furthermore, coherent surface acoustic wave sources with the same frequency and amplitude in two directions but opposite propagation directions are used. The surface acoustic wave in the first direction is generated by a surface acoustic wave generator, and the sound wave in the second direction is generated by reflection from an echo plate with a reflective surface. This method can reduce the number of sound wave generating devices and ensure that the frequencies and amplitudes of the waves in the two directions are the same, without the need for special adjustment.

[0012] Furthermore, the distance between the control surface sound source generator and the echo plate satisfies the standing wave resonance condition [L=(2n-1)]. [λ / 4, n=1,2,3,…], This distance setting can ensure the generation of standing waves.

[0013] Furthermore, the surface sound source generator produces surface sound source ultrasonic waves; the corresponding echo plate can completely reflect the surface sound source ultrasonic waves, generating reflected surface sound source ultrasonic waves.

[0014] Furthermore, both the surface acoustic generator and the echo plate are coupled to the gel electrophoresis carrier. This coupling configuration ensures the continuity of sound wave transmission and guarantees the concentration of low-concentration proteins on each band within the gel electrophoresis carrier.

[0015] Furthermore, a surface acoustic generator and an echo plate are set on the electrophoretic separation structure. The direction of the regular stripe standing wave generated by the surface acoustic generator and the echo plate is perpendicular to the electrophoretic direction of the electrophoretic separation structure. After the electrophoretic separation step is completed, the surface acoustic generator is turned on and works with the echo plate to concentrate proteins.

[0016] The present invention also discloses a device for enhancing the sensitivity of immunoblotting and immunofixation electrophoresis, which includes a surface acoustic generator, an echo carrier with a reflective surface, and a top cover.

[0017] Among them, the surface sound source generator is used to emit surface sound source ultrasonic waves.

[0018] The echo carrier includes an echo plate with a reflective surface, a mounting plate for a surface acoustic source generator on the side opposite to the echo plate, and two closed side plates perpendicular to the echo plate. The echo plate and the surface acoustic source generator are arranged opposite to and parallel to each other to ensure the formation of regular striped standing waves. The echo plate, the mounting plate, and the closed side plates surround a tank that contains the gel electrophoresis carrier.

[0019] The top cover is used to ensure that the entire concentration process takes place in a closed space.

[0020] The surface acoustic source generator emits a first-direction surface acoustic source ultrasonic wave, which is reflected by the echo plate to generate a second-direction surface acoustic source ultrasonic wave. The first-direction surface acoustic source ultrasonic wave and the second-direction surface acoustic source ultrasonic wave coherently superimpose to generate a regular stripe standing wave. The regular stripe standing wave passes through the gel electrophoresis carrier containing low concentrations of protein molecular bands that have been electrophoretically separated in a direction perpendicular to the electrophoretic separation.

[0021] Furthermore, the distance between the echo plate and the surface from which the sound wave is emitted by the surface sound source generator satisfies the standing wave resonance condition [L=(2n-1)]. [λ / 4, n=1,2,3,…], This distance can ensure the effect of standing waves, where L is the distance from the echo plate to the surface sound source generator, λ is the ultrasonic wavelength, and n is the harmonic sequence number of the resonance.

[0022] Furthermore, the device also includes an electrophoresis tank with four side plates around its perimeter and electrode plates. The closed side plates of the echo carrier are replaced by the side plates on both sides of the electrophoresis tank. The electrode plates are arranged perpendicularly to the echo plate and are located in the electrophoresis tank on both sides of the echo plate.

[0023] Furthermore, to simplify the processing of electrophoresis buffer, an electrophoresis buffer filling and discharging pipeline that can communicate with the electrophoresis tank is provided. The first end of the filling and discharging pipeline is an opening inside the tank, and the second end of the filling and discharging pipeline is a filling and discharging instrument connection port. This configuration facilitates the filling and discharging of electrophoresis buffer.

[0024] Furthermore, to ensure a more rational overall structural design, a main support is installed, with the electrophoresis tank mounted on it. The charging and discharging lines pass through the interior of the main support, ensuring the tank opening faces the electrophoresis tank. The second end of the charging and discharging lines extends out of the main support. Correspondingly, a circuit board, a power supply port, and a multi-functional switch controlling the electrophoresis separation and regular stripe standing wave concentration are installed within the main support. The main support ensures a neat and orderly instrument structure and smoother operation.

[0025] Technical effect By performing regular stripe standing wave concentration on the gel electrophoresis carrier directly in a direction perpendicular to the electrophoretic separation direction after electrophoretic separation, each low-concentration protein band obtained by electrophoresis can be further concentrated, so that the protein concentration reaches or is closer to the concentration at which a visible antigen-antibody reaction occurs. This provides the conditions for subsequent antibody binding and for the local signal peak intensity to reach or exceed the detection threshold in subsequent detection after membrane transfer (immunoblotting technology), which can greatly improve the detection rate of low-abundance target molecules.

[0026] The resonance condition for standing wave formation is satisfied by the distance between the surface sound source generator and the echo plate, as well as the distance between the surface sound source generator and the echo plate [L=(2n-1)]. Setting λ / 4, n=1,2,3,…] allows the generated regular fringe standing waves to produce nodes or antinodes more effectively, resulting in better concentration.

[0027] The effective operation of the above methods is ensured by setting up a special device.

[0028] By combining the electrophoretic separation step with the regular stripe standing wave concentration step into a corresponding device, time can be greatly saved and the operation steps can be simplified. The operation process only requires the discharge of electrophoretic solution to switch between the two steps.

[0029] By setting up corresponding charging and discharging pipelines to the main carrier, the charging and discharging of the electrophoresis solution becomes more convenient. The switching between the electrophoretic separation step and the regular stripe standing wave concentration step does not require moving the main body of the device, ensuring safer and more stable operation. Attached Figure Description

[0030] Figure 1 This is a schematic diagram of the exploded structure of the device in Example 2; Figure 2 This is a top view of the device in Example 2; Figure 3 This is a schematic diagram of the structure of the device in Example 2, showing the combination of the echo carrier and the single-hole support frame, with an electrode sheet on one side. Figure 4 This is a schematic diagram of the structure of the echo carrier and the single-hole support frame in the separated state of the device in Embodiment 2; Figure 5 This is a schematic diagram of the effect of the present invention on the results of immunofixation electrophoresis, wherein arrow ① indicates the direction of the electrophoretic separation electric field, and arrow ② indicates the direction of the standing wave concentration of the regular stripes.

[0031] Explanation of main figure symbols 1. Top cover; 2. Surface acoustic generator; 3. Echo carrier; 31. Echo plate; 32. Setting plate; 4. Single-hole support frame; 41. Support frame hole; 42. Side plate with platform; 421. Platform; 5. Electrode plate; 6. Electrophoresis tank; 7. Main support body; 71. Switch; 72. Power supply hole; 73. Charging and discharging pipeline; 731. Charging and discharging instrument connection port; 732. Tank opening. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of this application clearer, the technical solutions of this application will be clearly and completely described below in conjunction with specific embodiments and corresponding drawings. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of them. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0033] In this document, “illustrative” means “serving as an example, illustration or description”, and any illustration or implementation described herein as “illustrative” should not be construed as a more preferred or advantageous technical solution.

[0034] To keep the drawings concise, only the parts relevant to this application are shown schematically in each drawing, and they do not represent the actual structure of the product. In addition, to make the drawings concise and easy to understand, in some drawings, only one of the components with the same structure or function is shown schematically, or only one of them is labeled.

[0035] In this document, unless otherwise expressly specified and limited, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance; unless otherwise specified or explained, the term "multiple" refers to two or more; the terms "connected," "fixed," etc., should be interpreted broadly. For example, "connected" can be a fixed connection, a detachable connection, an integral connection, or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium. Those skilled in the art can understand the specific meaning of the above terms in this application according to the specific circumstances.

[0036] A standing wave is formed by the superposition of two coherent waves with the same frequency and amplitude in opposite directions. When these two waves superimpose, at some fixed locations, the vibration phases of the two waves are the same, and their amplitudes are enhanced, forming antinodes; at other fixed locations, the vibration phases of the two waves are opposite, and their amplitudes cancel each other out, forming nodes.

[0037] A volume wave is a wave that propagates in three-dimensional space within a medium, with its energy distributed across the cross-section of the propagation path.

[0038] A surface sound source is a type of sound source classified in acoustics according to its geometric shape. It refers to a sound source that vibrates in a planar form and whose phase and amplitude of the radiated sound waves are basically consistent at each point in its vibration plane. Its core characteristic is that the sound waves propagate outward in a planar direction.

[0039] Example 1. A method for enhancing the sensitivity of immunoblotting and immunofixation electrophoresis, comprising the following steps: S1: A regular striped standing wave generated by the superposition of ultrasonic waves from two coherent surface sound sources with the same frequency and amplitude but opposite propagation directions. S2: Regular stripe standing wave concentration of proteins is achieved by passing the gel electrophoresis carrier containing low concentrations of protein molecular bands after electrophoresis separation in a direction perpendicular to the electrophoretic separation direction. Surface acoustic waves propagate as volume waves within the carrier gel, overlapping to form stable standing waves. These standing waves have parallel planar nodes and antinodes within the gel carrier, which concentrate electrophoretically separated biomolecules within these nodes or antinodes. Ultimately, acoustic radiation pressure pushes protein molecules of varying densities towards the nodes or antinodes of the standing wave. Specifically, protein molecules with densities less than that of the gel medium are pushed towards the acoustic pressure antinodes, while those with densities greater than those of the gel medium are pushed towards the acoustic pressure nodes.

[0040] The acoustic radiation pressure of the regular striped standing wave described above concentrates low-concentration protein molecules of different concentrations, ensuring the proportionality of the antigen-antibody reaction.

[0041] Two coherent surface ultrasonic waves with the same frequency and amplitude in opposite directions are generated by a surface ultrasonic generator 2 in the first direction and a sound wave in the second direction by an echo plate 31 with a reflective surface. This method can reduce the number of sound wave generating devices and ensure that the frequencies and amplitudes of the two waves are the same, without the need for special adjustment.

[0042] The distance between the control surface sound source generator 2 and the echo plate 31 satisfies the standing wave resonance condition [L=(2n-1)]. [λ / 4, n=1,2,3,…], This distance setting can ensure the generation of standing waves.

[0043] The wavelength of ultrasound is shorter than the width of a single protein band. A more preferable approach is to use a wavelength less than or equal to half the width of a single protein band. This wavelength setting ensures that the protein band width can completely cover multiple nodes and antinodes of the standing wave, satisfying the requirement of concentration within the standing wave across multiple wavelength ranges. Specifically, a 5MHz ultrasound wave has a wavelength of 0.31mm in human tissue, resulting in an electrophoretic band width of approximately 3mm. By adjusting the frequency and amplitude, low-frequency ultrasound in pulse mode with a small amplitude is ultimately selected, effectively reducing heat generation during wave conduction in the gel and avoiding the risk of protein denaturation during the process.

[0044] In a more preferred embodiment, both the surface acoustic generator 2 and the echo plate 31 are coupled to the gel electrophoresis carrier. This coupling ensures the continuity of sound wave transmission and guarantees the concentration of low-concentration proteins on each band within the gel electrophoresis carrier. For example, the agarose gel itself can act as a coupling agent. Ultrasonic waves pass through the gel medium and contact the echo plate 31, satisfying the condition of half-wave loss due to propagation from a less dense medium to a denser medium, and the distance between the surface acoustic generator 2 and the echo plate 31 satisfies the standing wave resonance condition [L=(2n-1)]. [λ / 4, n=1,2,3,…], the result is the formation of standing waves in the gel medium.

[0045] In a more preferred embodiment, a surface acoustic generator 2 and an echo plate 31 are provided on the electrophoretic separation structure, with the orientation of the surface acoustic generator 2 and the echo plate 31 perpendicular to the electrophoretic direction of the electrophoretic separation structure. After the electrophoretic separation step is completed, the surface acoustic generator 2 is activated and works in conjunction with the echo plate 31 to concentrate proteins. This arrangement effectively avoids the risk of gel damage caused by device replacement and also greatly saves time.

[0046] A method for protein processing using the above-described approach first obtains a gel electrophoresis carrier after electrophoretic separation, placing the gel electrophoresis carrier with the electrophoretic separation direction perpendicular to the direction of the regular stripe standing wave. Then, a surface acoustic source generator 2 is activated to perform regular stripe standing wave concentration treatment on the gel electrophoresis carrier. The ultrasonic waves generated by the surface acoustic source generator 2 propagate through the gel electrophoresis carrier, coupling with the surface acoustic source. After reflection by the echo plate 31, a standing wave is formed in the gel carrier. After processing, subsequent antigen-antibody reaction steps such as immunoblotting and immunofixation electrophoresis are performed as needed.

[0047] Example 2 A device for enhancing the sensitivity of immunoblotting and immunofixation electrophoresis includes a surface acoustic generator 2, an echo carrier disk 3 with a reflective surface, and a top cover 1.

[0048] Among them, the surface sound source generator 2 is used to emit ultrasonic waves; The echo carrier 3 includes an echo plate 31 with a reflective surface, a mounting plate 32 for a surface acoustic source generator 2 on the side opposite to the echo plate 31, and two closed side plates perpendicular to the echo plate 31. The echo plate 31 is arranged opposite to and parallel to the surface acoustic source generator 2 to ensure the formation of regular striped standing waves. The echo plate 31, the mounting plate 32, and the closed side plates surround the tank that accommodates the gel electrophoresis carrier. The top cover 1 is used to ensure that the entire concentration process takes place in a closed space.

[0049] The surface acoustic source generator 2 emits a first-direction ultrasonic wave, which, after being reflected by the echo plate 31, generates a second-direction ultrasonic wave. The first-direction ultrasonic wave and the second-direction ultrasonic wave coherently superimpose to generate a regular striped standing wave. This regular striped standing wave passes through the gel electrophoresis carrier containing low-concentration protein molecule bands that have already been separated by electrophoresis in a direction perpendicular to the electrophoretic separation. During use, it is necessary to ensure that the echo plate 31 and the surface acoustic source generator 2 are positioned perpendicular to the electrophoresis direction; and both the echo plate 31 and the surface acoustic source generator 2 are coupled to the gel electrophoresis carrier.

[0050] The above-mentioned device setup ensures that the regular stripe standing waves generated by the surface acoustic source generator 2 and the echo plate 31 can concentrate low-concentration proteins on the gel electrophoresis carrier, ensuring the effectiveness of the subsequent antigen-antibody reaction, and thus enabling the detection of low-concentration proteins at the first time.

[0051] In a more preferred embodiment, the distance between the echo plate 31 and the sound wave emitting surface of the surface sound source generator 2 satisfies the standing wave formation resonance condition [L=(2n-1)]. [λ / 4, n=1,2,3,…], where L is the distance from the echo plate 31 to the surface sound source generator 2, λ is the ultrasonic wavelength, and n is the harmonic sequence number of the resonance. This distance ensures the standing wave effect.

[0052] In a more preferred embodiment, the device further includes an electrophoresis tank 6 with four side plates around its perimeter and electrode plates 5. The closed side plates of the echo plate 3 are replaced by the side plates on both sides of the electrophoresis tank 6. The electrode plates 5 are arranged perpendicularly to the echo plate 31 and are disposed within the electrophoresis tank 6 on both sides of the echo plate 31 to complete electrophoretic separation. This arrangement integrates the electrophoresis process and the regular stripe standing wave concentration process into a single device, simplifying operation. In practical use, the electrophoresis buffer can be poured into the electrophoresis tank 6 before electrophoretic separation, and the buffer can be poured out after separation before performing the regular stripe standing wave concentration process.

[0053] In a more preferred embodiment, in order to simplify the processing of electrophoresis buffer, an electrophoresis buffer filling and discharging pipeline 73 that can communicate with the electrophoresis tank 6 is provided. The first end of the filling and discharging pipeline 73 is an opening 732 inside the tank, and the second end of the filling and discharging pipeline 73 is a filling and discharging instrument connection port 731. This arrangement satisfies the convenience of filling and discharging electrophoresis buffer.

[0054] In a more preferred embodiment, to ensure a more rational overall structural design, a main support 7 is provided, with the electrophoresis tank 6 mounted on the main support 7. The charging / discharging pipeline 73 passes through the interior of the main support 7, causing the opening 732 of the tank to face the electrophoresis tank 6. The second end of the charging / discharging pipeline 73 extends out of the main support 7. A circuit board, a power supply port 72, and a switch 71 controlling the electrophoresis separation and regular stripe standing wave concentration are also provided within the main support 7. The main support 7 ensures a neat and orderly overall instrument structure and smoother operation.

[0055] In a more preferred embodiment, in order to ensure the gap between the bottom of the electrophoresis tank 6 and the echo tray 3 to ensure sufficient electrophoretic liquid to complete the electrophoretic separation action, and at the same time to ensure that the echo tray 3, which simultaneously performs the functions of electrophoretic separation and regular stripe standing wave concentration, is conveniently set up, a single-hole support frame 4 is also set between the electrophoresis tank 6 and the echo tray 3. The single-hole support frame 4 is provided with platform side plates 42 on both sides parallel to the echo plate 31, and also includes a perforated bottom plate with holes at the bottom that connects to the platform side plates 42. The holes in the perforated bottom plate are support frame holes 41, which communicate with the opening 732 inside the tank. The platform side plates 42 include a platform 421 in the middle position. The platforms 421 of the two platform side plates 42 are used to place the echo tray 3. The single-hole support frame 4 is set after the electrophoresis tank 6, and the platform side plates 42 are close to the inner side wall of the corresponding position of the electrophoresis tank 6. A more specific implementation is that the distance from platform 421 to the top of the side plate 42 is equal to the height of the echo carrier 3, ensuring that the entire top is flush after assembly. The single-hole carrier 4 provides support while ensuring that the electrophoresis solution is fully immersed in the gel, and also ensures that the electrophoresis solution fills the space between the electrode sheet 5 and the gel, releasing all the functions of the electrophoresis solution, including maintaining a stable pH environment, providing ion carriers, forming a closed circuit, maintaining appropriate ion strength, balancing heat generation and mobility, and serving as a heat exchange medium to help dissipate heat—essential prerequisites for successful electrophoresis.

[0056] The operating principle of the device is as follows: First, the surface acoustic generator 2 is placed inside the echo tray 3, and the prepared gel electrophoresis carrier is placed inside the echo tray 3. Then, the echo tray 3 is placed inside the single-well support 4, and the single-well support 4 is placed inside the electrophoresis tank 6. The electrode sheet 5 is placed vertically on both sides of the echo plate 31 in the electrophoresis tank 6 and connected to the power supply. Then, the electrophoresis space is sealed using the top cover 1. The electrophoresis separation step is started by turning on switch 71. After the electrophoresis separation is completed, the electrophoresis separation step is turned off. Then, the regular stripe standing wave concentration step is started by turning on switch 71. Ultrasonic waves emitted by the surface acoustic generator 2 are reflected by the echo plate 31, causing the two directional waves to form regular stripe standing waves. Under the action of the regular stripe standing waves, the proteins are concentrated. After the concentration is completed, the power supply is turned off, the top cover 1 is opened, and then the subsequent antigen-antibody reaction is carried out. The reference figure shows the various protein bands on the gel electrophoresis carrier after color development.

[0057] The above description is merely a specific embodiment of this application. Under the guidance of the above teachings, those skilled in the art can make other improvements or modifications based on the above embodiments. Those skilled in the art should understand that the above specific description is only to better explain the purpose of this application, and the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A method for enhancing the sensitivity of immunoblotting and immunofixation electrophoresis, characterized in that, The steps include, S1: A regular striped standing wave generated by the superposition of ultrasonic waves from two coherent surface sound sources with the same frequency and amplitude but opposite propagation directions. S2: Regular stripe standing wave concentration of proteins is achieved by passing the gel electrophoresis carrier containing low concentrations of protein molecular bands after electrophoresis separation in a direction perpendicular to the electrophoretic separation direction. The pressure of acoustic radiation is used to push protein molecules of different densities toward the nodes or antinodes of a standing wave; protein molecules with a density less than that of the gel medium are pushed toward the antinodes, while protein molecules with a density greater than that of the gel medium are pushed toward the nodes.

2. The method according to claim 1, characterized in that, The first direction surface sound source ultrasonic wave with the same frequency and amplitude in two directions and opposite propagation directions is generated by a surface sound source generator, and the second direction sound wave is generated by reflection by an echo plate with a reflective surface.

3. The method according to claim 1, characterized in that, The distance between the control surface sound source generator and the echo plate satisfies the standing wave resonance condition: L=(2n-1) λ / 4.

4. The method according to claim 1, characterized in that, The wavelength of the ultrasound is less than the width of a single protein band; preferably, the wavelength of the ultrasound is less than or equal to half the width of a single protein band.

5. The method according to claim 1, characterized in that, Both the surface acoustic source generator and the echo plate are coupled to the gel electrophoresis carrier.

6. The method according to claim 1, characterized in that, A surface acoustic generator and an echo plate are set on the electrophoretic separation structure. The direction of the regular stripe standing wave generated by the surface acoustic generator and the echo plate is perpendicular to the electrophoretic direction of the electrophoretic separation structure. After the electrophoretic separation step is completed, the surface acoustic generator is turned on and works with the echo plate to concentrate proteins.

7. A device for enhancing the sensitivity of immunoblotting and immunofixation electrophoresis, characterized in that, It includes, A surface acoustic generator is used to emit surface acoustic ultrasonic waves. The echo carrier includes an echo plate with a reflective surface, a mounting plate for a surface acoustic source generator on the side opposite to the echo plate, and two closed side plates perpendicular to the echo plate. The echo plate and the surface acoustic source generator are arranged opposite to and parallel to each other to ensure the formation of regular striped standing waves. The echo plate, the mounting plate, and the closed side plates surround a tank that contains the gel electrophoresis carrier. The top cover is used to ensure that the entire concentration process takes place in a closed space; The surface acoustic source generator emits a first-direction surface acoustic source ultrasonic wave, which is reflected by the echo plate to generate a second-direction surface acoustic source ultrasonic wave. The first-direction surface acoustic source ultrasonic wave and the second-direction surface acoustic source ultrasonic wave coherently superimpose to generate a regular stripe standing wave. The regular stripe standing wave passes through the gel electrophoresis carrier containing low concentrations of protein molecular bands that have been electrophoretically separated in a direction perpendicular to the electrophoretic separation.

8. The apparatus according to claim 7, characterized in that, The distance between the echo plate and the surface from which the sound waves are emitted by the surface sound source generator is an integer multiple of the ultrasonic wavelength of the surface sound source.

9. The apparatus according to claim 7, characterized in that, The device also includes an electrophoresis tank with four side plates around it and electrode plates. The closed side plates of the echo carrier are replaced by the side plates on both sides of the electrophoresis tank in the corresponding direction. The electrode plates are arranged perpendicularly to the echo plate and are located in the electrophoresis tank on both sides of the echo plate.

10. The apparatus according to claim 9, characterized in that, The device also includes an electrophoresis buffer filling and discharging line that can communicate with the electrophoresis tank. The first end of the filling and discharging line is an opening inside the tank, and the second end of the filling and discharging line is a filling and discharging instrument connection port. Preferably, a main carrier is provided, the electrophoresis tank is set on the main carrier, and the charging and discharging pipeline passes through the inside of the main carrier so that the opening inside the tank faces the electrophoresis tank; the second end of the charging and discharging pipeline extends out of the main carrier; a circuit board is provided in the main carrier, and a power supply hole and a switch for controlling the electrophoresis separation and the regular stripe standing wave concentration are also provided. Preferably, a single-hole support frame is further provided between the electrophoresis tank and the echo tray. The single-hole support frame is provided with platform side plates on both sides parallel to the echo plate, and also includes a perforated bottom plate with holes at the bottom connecting the platform side plates. The platform side plates include a platform in the middle position, and the platforms of the two platform side plates are used to place the echo tray. The single-hole support frame is provided behind the electrophoresis tank, and the platform side plates are close to the inner side wall of the corresponding position of the electrophoresis tank. Preferably, the distance from the platform to the top of the side plate is equal to the height of the echo carrier, so as to ensure that the entire top is flush after assembly.

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